An important part of the development of a protein therapeutic is the preparation of the protein in a stabilised form that can be stored over extended periods without loss of activity so that the dose and activity of the protein therapeutic can be carefully controlled.
Polypeptides can lose biological activity as a result of physical instabilities, including denaturation and formation of soluble and insoluble aggregates, and a variety of chemical instabilities, such as hydrolysis, oxidation, and deamidation. Stability of polypeptides in liquid pharmaceutical formulations can be affected, for example, by factors such as pH, ionic strength, temperature, repeated cycles of freeze-thaw, and exposure to mechanical shear forces such as occur during processing. Aggregate formation and loss of biological activity can also occur as a result of physical agitation and interactions of polypeptide molecules in solution and at the liquid-air interfaces within storage vials. Further conformational changes may occur in polypeptides adsorbed to air-liquid and solid-liquid interfaces during compression-extension of the interfaces resulting from agitation during transportation or otherwise. Such agitation can cause the protein to entangle, aggregate, form particles, and ultimately precipitate with other adsorbed proteins. For a general review of stability of protein pharmaceuticals, see, for example, Manning et al. (1989) Pharm. Res. 6: 903-918, and Wang and Hanson (1988) J. Parenteral Sci. Tech. 42: S14.
The development of multimer protein therapeutics presents additional challenges especially if the integrity of the multimer protein is essential for the efficacy of the therapeutic end product. In such cases, not only is it necessary to ensure that the protein is maintained without loss of activity and without protein precipitation or aggregation but it is also important to check if the integrity of the multimer protein has been maintained in the therapeutic end product.
To date, workers in the field of stabilisation of multimer proteins, such as a bacterial ADP-ribosylating exotoxin class proteins (bAREs) which are organised as A:B multimers have been faced with at least two different types of problems when working with such proteins. In particular, the bARE proteins may lose their biological activity: (i) as a result of physical instabilities including denaturation and formation of soluble and insoluble aggregates; and/or (ii) by the partial or complete dissociation of the bARE protein into its A and B subunit forms.
Sometimes it may be easy to see when a protein, such as a multimeric protein precipitates out of solution because aggregate or crystalline particles may form. On the other hand, it is not so easy to determine if a multimeric protein has dissociated either partially or completely into its subunit forms because, for example, a protein assay, will not differentiate over intact and dissociated forms of the multimeric protein.
To date, no information has been available on how to measure the integrity of bARE proteins multimers without loss of the integral multimer structure. Current analytical methods used to characterize multimeric bARE class proteins, including electrophoresis, immunoblotting, mass spectrometry and amino acid analysis, are unable to distinguish between the integral multimeric structure and the separate dissociated subunit forms. This failure to differentiate between integral and dissociated bARE proteins is due to the fact that the current analytical techniques require the dissociation of the A and B subunit forms and so do not maintain the structural organisation of the integral multimeric bARE molecule. Neither do these techniques permit the quantitation of the integral bARE molecule relative to the dissociated subunit molecule. Therefore, current analytical methods are neither useful for studying the dissociation (or loss of integrity) of the multimeric bARE protein over time nor for looking for ways to stabilise the multimeric bARE protein. In addition, even when traditional separation methods, such as gel filtration methods are used, which do not require the dissociation into monomeric subunit forms, these methods are not very effective as they do not permit a good resolution of the different subunit forms because of extremely close retention times.
As a result, up until now, a reliable method of determining whether or not a particular sample of a bARE molecule is present in an intact or dissociated form has not been available to workers in the field of bARE molecules. Such a method would be useful not only for: (i) distinguishing between the integral multimeric bARE structure and separate, dissociated A and B subunits but would also be useful for (ii) investigating and determining the conditions required for stability of a bARE class protein including the identification of effective stabilizing agents. Methods of evaluating, achieving and quantifying the stability of bARE class proteins would be very useful for (iii) the development of appropriate formulations for bARE class proteins for stable storage and delivery of the bARE protein, either alone or, for example, as part of a composition, such as an immunogenic (e.g., vaccine) composition.
As mentioned above, another major obstacle that must be overcome in the use of protein-based pharmaceuticals, such as those including a bARE protein as a therapeutic agent is the loss of pharmaceutical utility that may result from its instability in pharmaceutical formulations. The stabilization of polypeptides in pharmaceutical compositions remains an area in which trial and error plays a major role (reviewed by Wang (1999) Int. J. Pharm. 185:129-188; Wang and Hanson (1988) J. Parenteral Sci. Tech. 42:S3-S26). Excipients that are added to polypeptide pharmaceutical formulations to increase their stability include buffers, sugars, surfactants, amino acids, polyethylene glycols, and polymers, but the stabilizing effects of these chemical additives may vary depending on the protein. Physical instabilities that threaten polypeptide activity and efficacy in pharmaceutical formulations include denaturation and formation of soluble and insoluble aggregates. Some of these changes are known to lead to the loss or reduction of the pharmaceutical activity of the bARE protein of interest. In other cases, the precise effects of these changes are unknown, but the resulting degradative products are still considered to be pharmaceutically unacceptable due to the potential for undesirable side effects.
Aggregate formation by a polypeptide such as a bARE molecule during storage of a pharmaceutical composition can adversely affect biological activity of that polypeptide, resulting in loss or reduction of the pharmaceutical activity or therapeutic efficacy of the pharmaceutical composition. Furthermore, aggregate formation may cause other problems such as blockage of tubing, membranes, or pumps when the functional stability of a bARE protein is determined using an analytical system, such as a chromatographic method or when the bARE protein based pharmaceutical composition is administered using an infusion system. In addition, injection of a pharmaceutical composition comprising the aggregated form of a protein has the potential for generating an immunogenic reaction to the aggregated protein.
Consequently, there is also a need to: (i) find ways of determining the stability of a bARE protein over time; (ii) identify stabilisers that can improve the stability of a bARE protein over time; and (iii) provide bARE compositions which are stable on storage and which have a prolonged activity over time.